1. Field of the Invention
The invention relates generally to the exportation of heterologous polypeptides to discrete regions of a host cell in which it is expressed, to nucleic acid sequences encoding exportation polypeptides, to the preparation of membrane embedded epitopes of immunogenic antigens, and to vectors constructed with selected exportation sequences. More particularly, localized expression of polypeptides may be obtained by providing exportation signals encoded by segments of the disclosed nucleic acids that provide for exportation of expressed heterologous polypeptides to the inner membrane/periplasmic space or the outer membrane surface of a host cell.
2. Description of Related Art
Recombinant gene technology has been extensively investigated in the context of expression of foreign proteins in host cells which harbor recombinant genes, typically bacterial host cells. Such expression is desirable for producing high value proteins, immunogenic polypeptides, and in obtaining hybrid proteins that are otherwise difficult to synthesize.
Of particular interest is vaccine development. It is potentially feasible to prepare protective vaccines from epitopes of known antigens of eukaryotic, viral or prokaryotic pathogens by taking advantage of the synthetic capacities of transformed host cells. Examples include tumor specific proteins which might be expressed and utilized to stimulate an immune response. Oral vaccines have stimulated research because of the ease of administration and, more importantly, in some instances the unsatisfactory protection afforded from parenteral injection. Vaccination against cholera, for example, gives short-term protection, thus provoking developmental work toward an oral vaccine that would presumably stimulate mucosal intestinal immunity more efficiently (Sanchez et al., 1990).
Salmonella strains are being studied experimentally as particularly attractive candidates for producing oral live vaccines. Attenuated strains have been shown to elicit immune responses in several animal species (Strugnell et al., 1990) and apparently can be highly immunogenic in the host. Humoral antibody responses including local secretory antibody and cellular immune responses have been observed after oral intake (Dougan et al., 1986). Attenuated mutants have been identified via screening procedures such as TnphoA mutagenesIS, which exclude elimination of mutations in nonsecreted proteins (Miller et al., 1989). However, TnphoA methods only indicate assessment of integration of the transposon into a gene for a secreted or cytoplasmic protein.
Protein expression systems have been developed from Salmonella strains. A cloning vector useful for integrating DNA into the aroC gene on Salmonella chromosomes was used to direct expression of heterologous antigens such as tetanus toxin fragment C and Treponema pallidum lipoprotein (Strugnell et al., 1990). In some cases, heterologous polypeptide gene products orally administered have elicited a serum antibody response, as for example, the cholera toxin B subunit protein expressed from a recombinant Yersinia enterocolitica strain (Sory and Cornelis, 1990). Unfortunately, while antibodies were detected in sera of challenged mice, the response was variable and was directed toward polymeric forms of cholera toxin B.
It is recognized that cytoplasmic proteins may not produce a high immunogenic response and heterologous proteins from recombinant DNA molecules expressed cytoplasmically often exhibit a diminished antibody reactivity (Sanches, Et. Al., 1990). Thus surface expressed epitopes of bacteria are expected generally to elicit the greatest humoral response; however, factors controlling surface expression of heterologous proteins have not been defined and there is no way to assure that any given fusion protein will localize to a host cell membrane surface.
Vaccines are the most cost effective medical intervention known to prevent disease. However, effective vaccines are available for relatively few diseases. Successful immunization against infectious organisms often requires a multicomponent host immune response against a variety of antigenic determinants. Orally administered vaccines, especially live attenuated vaccines, induce specific cell-mediated effector responses and elicit secretory IgA (sIgA) responses. SIgA is important because of its effectiveness at mucosal surfaces. SIgA production and cell effector responses are mediated through the delivery of antigens to gut-associated lymphoid tissue (GALT). Stimulation of GALT can lead to effective cell and humoral defense at all mucosal surfaces and provide systemic protection (1,2).
To deliver antigens to GALT, investigators have developed avirulent and virulence-attenuated Salmonella stains. Aromatic dependent (aroA (3)), phoP (4), galE (5), and cya/crp (6) Salmonella mutants have been reported to interact with GALT in the lamina propria and stimulate an immune response. While it is clearly desirable to use avirulent Salmonella strains as carriers for plasmids which express protective antigens of other pathogens on their surface, it is clear that improvements are needed to develop protective vaccines based on this system.
The use of attenuated Salmonella strains to express heterologous antigens and stimulate GALT is being extensively investigated. In some studies, detectable levels of specific mucosal and serum antibodies to the heterologously expressed antigen have been observed (7-10). However, in general results with most antigens have been variable.
It is generally believed that the export of heterologous epitopes to the Salmonella cell surface enhances their immunogenicity (11). Investigators have used recombinant DNA methods to express heterologous epitopes as inserts in Salmonella flagellin (9) and the 1amb encoded polypeptide of E. coli (10). In these studies, a significant antibody response to the heterologous surface-expressed epitopes was observed. A limitation of these systems is the relatively small number of epitopes which can be inserted into the 1amb and flagellin genes. This is important as single (or few) epitopes may not result in the broad-based immune response which characterizes today's most successful vaccines.
There is clearly a need to develop effective systems to elicit antibody response and in particular to provide methods of exporting heterologous polypeptides to the surface of appropriate host cells. Antigenic peptides expressed on bacterial host cell surfaces may be significant in developing vaccines to such important antigens as cholera B subunit toxin and HIV antigens.